Anti-HPV E7 antibodies

ABSTRACT

The present invention relates to monoclonal anti-HPV (human papillomavirus) E7 antibodies capable of specifically recognizing an epitope of the C-terminal or the N-terminal region of a HPV E7 protein, diagnostic compositions and kits comprising said antibodies as well as methods for immunohistochemical and ELISA-based diagnosis of HPV infections utilizing said antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/578,985 filed on Aug. 14, 2012, which is a 371 U.S. National Stage ofInternational Application No. PCT/EP2011/000728, filed Feb. 16, 2011,which claims priority to European Patent Applications No. EP 10001569.2,filed Feb. 16, 2010 and EP 10011978.3, filed Sep. 30, 2010. Thedisclosures of the above applications are incorporated herein byreference.

DESCRIPTION

The present invention relates to monoclonal anti-HPV (humanpapillomavirus) E7 antibodies capable of specifically recognising anepitope of the C-terminal or the N-terminal region of a HPV E7 protein,diagnostic compositions and kits comprising said antibodies as well asmethods for immunohistochemical and ELISA-based diagnosis of HPVinfections utilizing said antibodies.

Sexually transmitted infections with human papillomaviruses (HPVs) arethe main etiological factor for cervical cancer. Over forty, inparticular over thirty-five HPV genotypes, that can infect epithelialsquamous and glandular cells in the cervical mucosa, have beendescribed. On the basis of epidemiological and biochemical data, thedouble stranded DNA HPVs are divided into high-risk HPVs associated withsquamous intraepithelial lesions with a high potential for progressionto invasive squamous cell carcinoma, and low-risk HPVs associated withbenign hyperplasia. Infections by high-risk genotypes have been detectedin almost all neoplastic lesions of the cervix and at least 15 high-risktypes of HPV have been associated with cervical cancer. HPV 16 is themost prevalent genotype worldwide with an incidence in squamous cellcarcinoma of approximately 55%, followed by HPV 18 (approximately 17%)and HPV 45 (4-9%).

The persistence of oncogenic HPV is necessary for the development ofcervical precancer and cancer. However, the factors that determine viralpersistence and tumorigenic progression are not fully understood. Theinitial events of cervical carcinogenesis after viral infection dependon the fact that high-risk HPV types undergo specific changes thatabrogate the transcriptional control of viral gene expression in theinfected keratinocytes. Inactivation of these cellular control functionspermits deregulated transcription of the early viral genes E6 and E7,thereby triggering cell proliferation, inhibition of apoptosis,reprogramming of differentiation and chromosomal instability. Thesechanges can support the integration of episomal HPV genomes intochromosomes of the host cell, and contribute to further overexpressionof the viral genes E6 and E7, resulting in an increase of the E7oncoprotein levels during early steps of cervical carcinogenesis.

That the viral oncoproteins E6 and E7 are crucial during carcinogenesiswas further proven by the fact that high-risk E7 protein, in cooperationwith high-risk E6, can efficiently immortalize human primarykeratinocytes in vitro. Moreover, the consistent overexpression of theE6 and E7 oncogenes is required to induce and to maintain thetransformed phenotype of cervical cancer cells.

Thus, overexpression of E7 oncoproteins of carcinogenic HPV types is acharacteristic feature of cervical cancer. This conclusion is supportedby a recent study where affinity-purified antibodies against high-riskHPV E7 proteins were used to detect the E7 oncoproteins of HPV 16, 18and 45 in biopsies from invasive cervical squamous cell carcinomapatients (Ressler et al., 2007, Clin Cancer Res, 13:7067-7072),indicating that the high-risk E7 oncoproteins of these major high-riskHPV types are expressed continuously in invasive cervical carcinoma.

At present, clinical cervical cancer screening is mainly based on thecytological assessment of cells contained in cervical smears, referredto as Pap Smears (or Papanicolaou test; Papanicolaou, 1942, Science,95:438-439), that are routinely taken from women participating inscreening programs. The result of the cytological analysis is dependenton detection of abnormal cells by experienced pathologists. Although theimplementation of this simple and efficient assay has helped toconsiderably reduce the mortality of cervical cancer, Pap Smeardiagnosis is still characterized by a high rate of false-positive andfalse-negative results (Foucar, 2005, Semin Diagn Pathol,22(2):147-155).

In an alternative approach, detection of high-risk HPV DNA has beenintroduced into clinical practice. In this setting, a sensitive DNAdetection assay (e.g., Hybrid capture 2™; Digene Inc., USA) is appliedto determine the presence of high-risk HPV DNA in patient samples.Although this assay is well suited to detect infections bypapillomaviruses of the high-risk type, it cannot discriminate betweentransient infections, which spontaneously regress in most cases, and theonset of cervical cancer. More recently, diagnostic systems have beendeveloped that detect the presence of E6/E7 mRNA in cervical smears, asa surrogate marker for the expression of the viral oncoproteins E6 andE7. However, it has been shown that the level of E6/E7 mRNA is notsignificantly changing during carcinoma progression, suggesting that RNAdetection may not be the best tool for cervical cancer screening (Hafneret al., 2008, Oncogene, 27(11):1610-1617). In accordance with thisnotion, the underlying HPV infection could not be detected by PCRanalysis in all examined tumors by Ressler et al., 2007.

Therefore, the detection of E7 protein seems to be the superiordiagnostic tool. Various antibodies are already known in the art, butthey either display low sensitivity or specificity or are described tocross-react with the E7 proteins of various HPV types. Also, aspolyclonal antibodies can only be produced in a limited quantity by oneanimal, there are batch-to-batch differences. The difficulty to producehighly specific and sensitive monoclonal antibodies against E7 proteinsis mainly due to the low immunogenicity of E7 proteins.

WO 07/059492 for example, discloses methods, assays and kits for thedetection of HPV DNA or proteins, employing inter alia monoclonal andpolyclonal antibodies against the HPV 16 E7 protein, capable of reliablydetecting the rather high amount of around 1 μg of recombinant HPV 16 E7protein.

Accordingly, the technical problem underlying the present invention isto overcome the aforementioned difficulties and to provide highlyspecific and more sensitive means for a cost-efficient, rapid andreliable diagnosis of high-risk HPV infections.

The present invention solves its underlying problem according to theteaching of the independent claims. Accordingly, the present inventionprovides monoclonal antibodies against the E7 proteins of high-risk HPVtypes, in particular provides a monoclonal anti-HPV (humanpapillomavirus) E7 antibody, which is capable of specificallyrecognising an epitope, in particular a conformation-specific epitope,of the C-terminal region of a HPV E7 protein.

The term “antibody” as employed herein preferably refers to a fullimmunoglobulin, like an IgG, IgA, IgM, IgD or IgE but in anotherembodiment may also refer to a fragment of an antibody, like an F(ab),F(abc), Fv, Fc or F(ab)₂ or a fused antibody, a fused antibody fragmentor another derivative of the antibody of the present invention as longas it still displays the specificity and sensitivity of the fullimmunoglobulin of the present invention.

The term “specificity” or “specifically recognising/binding” isunderstood to refer to the property of an antibody to specificallyrecognise or bind to preferably only one specific epitope and preferablyshows no or substantially no cross-reactivity to another epitope.Preferably “specificity” or “specifically recognising/binding” meansthat the antibody specifically recognises or binds to preferably onlyone specific epitope and preferably shows no or substantially nocross-reactivity to another epitope, wherein said epitope is unique forone protein, such that the antibody shows no or substantially nocross-reactivity to another epitope and another protein.

The term “sensitivity” as employed herein refers to the detection limitof an antibody, which is preferably low, i.e. at least below aconcentration of 1 μg of the protein to be detected.

In general, the term “epitope” refers to an antigenic region of a givenprotein which consists of 4 to 50, preferably 5 to 15, preferably 8 to11 amino acids, or more. This antigenic region or epitope isspecifically recognised by the antigen binding site(s) of the antibody.

In the context of the present invention, the term “C-terminal region” isunderstood to refer to the at most 30, 35, 40, 45, 50, 55 or 60C-terminal amino acids of the HPV E7 protein.

In a preferred embodiment, the monoclonal anti-HPV E7 antibodyspecifically recognises an epitope of the zinc finger domain of theC-terminal region of the HPV E7 protein, preferably said epitope is aconformational epitope.

The monoclonal antibodies according to the present invention arepreferably used to detect native, i.e. non-denatured HPV E7 proteins.

In the context of the present invention, the term “conformationalepitope” refers to a three-dimensional epitope which the antibodyspecifically recognises or binds to as opposed to a linear epitope.Linear epitopes are determined solely by the amino acid sequence,whereas conformational epitopes are determined by the three-dimensionalsurface features, i.e. the tertiary structure. Therefore, the aminoacids of a conformational epitope defining the antigenicity of a proteindo not necessarily have to be consecutive amino acids of the protein butcan be distant from each other in the primary protein sequence to formthe conformational epitope, only if the protein is folded in itstertiary structure. Mostly, such conformational epitopes cannot berecognised by the antibody, if the protein is denatured, i.e. not foldedinto its tertiary structure.

HPV E7 proteins contain a zinc finger domain in their C-terminal part,which forms a highly ordered rigid structure, mainly due to 4 cysteinresidues. These cystein residues coordinate zinc and thereby represent aconformational epitope. Surprisingly, it has been found that themonoclonal antibodies according to the present invention which arecapable of recognising or binding to such a conformational epitope, areable to detect a HPV E7 protein in a highly specific and highlysensitive manner.

In a preferred embodiment, the monoclonal antibody is capable ofspecifically recognising an epitope of the C-terminal region, preferablythe zinc finger domain, of the E7 protein of HPV 16 subtypes, inparticular HPV types 16, 31, 33, 35, 52, 58 or of HPV 18 subtypes, inparticular HPV types 18, 39, 45, 59, 68, 70 or of both HPV 16 and 18subtypes. Preferably, the recognised epitope is a conformationalepitope. Preferably, the recognised epitope comprises the amino acidsequence set forth in SEQ ID No. 1 which is a common epitope sequencemotif present in epitopes of the present invention. The epitope motifcharacterised by SEQ ID No. 1 represents a consensus epitope motif forHPV 16 and 18 subtypes and is: Val Cys Pro Xaa Cys, with Xaa being anyamino acid.

The term “consensus epitope motif” as employed herein refers to specificparts of an epitope which are shared by the E7 proteins of HPV 16 and/or18 subtypes. Despite sharing such a consensus epitope motif, theC-terminal regions of HPV 16 and/or 18 subtypes, in particular thespecific epitopes contained therein and containing this epitope motifare, due to specific amino acid sequences and/or a specific conformationof the amino acid sequence, substantially different enough, so thatspecific antibodies are able to specifically recognise a particular E7protein from a specific HPV type, i.e. are capable of identifyingspecifically one particular HPV type.

In a preferred embodiment of the present invention, the monoclonalantibody is capable of specifically recognising an epitope of theC-terminal region, preferably the zinc finger domain, of HPV 16subtypes, in particular 16, 31, 33, 35, 52 and 58. Preferably, therecognised epitope is a conformational epitope. Preferably, therecognised epitope comprises the amino acid sequence set forth in SEQ IDNo. 2 which is a common sequence motif present in epitopes of the HPV 16subtypes. The epitope motif characterised by the amino acid sequence ofSEQ ID No. 2 represents a consensus epitope motif specific for HPV 16subtypes, and is: Gly Xaa Xaa Xaa Xaa Val Cys Pro Xaa Cys, with Xaabeing any amino acid.

In a particularly preferred embodiment of the present invention, themonoclonal antibody is capable of specifically recognising the E7protein of HPV 16. Preferably, the recognised epitope comprises,preferably consists of, the amino acid sequence set forth in SEQ ID No.3. The epitope characterised by the amino acid sequence of SEQ ID No. 3represents a specific conformational epitope of the zinc finger domainin the C-terminal part of the HPV 16 E7 protein, and is: Gly Thr Leu GlyIle Val Cys Pro Ile Cys Ser Gin Lys. These amino acids representpositions 85 to 97 of the HPV 16 E7 protein. A further particularlypreferred embodiment of the conformational epitope is represented by theamino acid sequence of SEQ ID No. 7 and is: Thr Leu Gly Ile Val Cys ProIle Cys Ser Gin Lys Pro. These amino acids represent positions 86 to 98of the HPV 16 E7 protein. The amino acid sequence of SEQ ID No. 6represents a specifically preferred embodiment of the conformationalepitope of SEQ ID No. 3 and 7 and is: Thr Leu Gly Ile Val Cys Pro IleCys Ser Gin Lys. These amino acids represent positions 86 to 97 of theHPV 16 E7 protein. All of these epitope sequences comprise two of thefour cystein residues which coordinate the zinc ion of the zing fingerdomain in two CXXC motifs. Epitope mapping using microarrays with HPV 16E7 derived synthetic 13 mer peptides identified this epitope to berecognised by the monoclonal antibody of the present invention veryspecifically (see FIG. 10). An alignment of HPV E7 protein sequencessuggested that especially the amino acids upstream and downstream of thehighly conserved 90VCPXC94 motif within the epitope of SEQ ID No. 3, 6and 7 provide the specific recognition of the HPV 16 E7 protein by themonoclonal antibody of the present invention.

A mutational analysis showed that peptides containing a point mutationin the zinc coordination site could not be detected by the monoclonalantibody against the E7 protein of HPV 16, whereas deletion mutants ofother regions could be readily detected. This clearly indicates that anintact zinc finger domain, representing a conformational epitope, isnecessary for the specific recognition of HPV 16 E7 protein by themonoclonal antibody according to the present invention. This is bestillustrated by the inability of the monoclonal antibody against HPV 16E7 of the present invention to recognise the HPV 16 E7 mutant C58G,wherein the cystein residue at position 58 is changed to glycine,containing the sequence required to recognise the linear epitope but haslost the ability to provide this epitope in a conformational zinc fingerdomain.

In accordance with the conformational nature of the epitope of SEQ IDNo. 3 and 7, in particular SEQ ID No. 6, the monoclonal antibodyrecognising this epitope has to be used in much higher concentrations(e.g. 15 μg/ml to detect 10 ng of purified protein) in applications likeWestern blot or protein recognition in cell lysates, where the proteinsare denatured, than known polyclonal antibodies against HPV 16 E7.

This epitope, preferably C-terminal conformational epitope, lies withina region of the E7 protein usually displaying low immunogenicity.Surprisingly, it could be shown that the monoclonal antibody recognisingthe epitope as given in SEQ ID No. 3 and 7, in particular SEQ ID No. 6,is able to specifically detect the E7 protein of HPV 16 in a highlysensitive manner and does not cross-react with E7 proteins of other HPVtypes, preferably in non-denaturing applications. In particular, themonoclonal antibody against HPV 16 E7 did not cross react with the E7proteins of HPV 18 or HPV 45, when these proteins were overexpressed inU-2OS cells. Also, the monoclonal antibody against HPV 16 E7 stronglydetected endogenous E7 protein of HPV 16 positive CaSki cells inimmunofluorescence experiments. In contrast, no signal was observed inuntransfected U-2OS cells or in HPV 18 positive HeLa cells. To moreprecisely characterise the cross reaction of the monoclonal antibodyagainst HPV 16 E7 an ELISA assay using E7 proteins of different HPVtypes was employed. Also, no cross reactivity was found against the E7proteins of other HPV types such as other high risk HPV genotypes andthe two most common low risk HPV types, HPV 6 and HPV 11.

Further, preincubation of the monoclonal antibody against HPV 16 E7 withthe purified HPV 16 E7 protein prevented the detection of cellassociated HPV 16 E7, since the antibody was already specifically boundto the preincubated HPV 16 E7 protein.

Compared to the prior art, the present monoclonal antibody is at least20 times, in particular at least 100 times, more sensitive with no lossof specificity. As infections with HPV 16 account for over 50% of cervixcarcinoma cases worldwide, the highly specific antibody according to thepresent invention is suitable to serve as a highly effective andreliable diagnostic tool. Advantageously, it could be shown that themonoclonal anti-HPV 16 E7 antibody according to the present invention isable to detect low amounts such as 1 μg, in particular 500 fg, of HPV 16E7 protein in an ELISA-based assay (enzyme-linked immunosorbant assay),whereas the antibodies disclosed in WO 07/059492 could only detectconcentrations of HPV 16 E7 protein of about 1 μg. The monoclonalanti-HPV 16 E7 antibody according to the present invention can thereforebe used in much lower concentrations, for example 10 μg/μl, whichrenders its application as a diagnostic tool also more cost-effective.

Using the present monoclonal antibody against HPV 16 E7 it could also beshown that subcellular localisation of HPV 16 E7 protein varies duringcell cycle between predominantly cytoplasmic and predominantly nuclear.

It has been shown that E7 protein levels increase from undetectable tosubstantial levels during carcinogenesis. With the highly sensitivemonoclonal antibodies according to the invention it is possible todetect not only tumor cells of squamous cell carcinoma andadenocarcinomas, but also cells of high-grade precancerous cervicalintraepithelial neoplasia and carcinoma in situ (CIN III and CIS) aswell as cervical intraepithelial glandular neoplasia grade III andadenocarcinoma in situ (CIGN III and AIS), which were reported to have ahigh potential to progress to invasive cancer. This might advantageouslypermit early diagnosis of high-grade precancerous CIN III, CIS, CIGNIII, ACIS, and AIS before their progression to malignant tumors, as themonoclonal antibodies according to the present invention candiscriminate between low grade precancerous lesions of squamous cell andglandular origin (CIN I-II, CIGN I-II) on the one hand, and high-gradeprecancers (CIN III, CIS, CIGN III) and cancers (CxCa, AdCa) of squamouscell and glandular origin on the other hand. Thus, the monoclonalantibodies according to the present invention can serve as a new tool toidentify high-grade precancers and cancers of squamous cell andglandular origin.

Thus, the present invention preferably relates to a monoclonal antibodyspecifically recognising an epitope comprising the amino acid sequenceof SEQ ID No. 1, 2, 3 and 4, preferably comprising or consisting of SEQID No. 3 and 7, in particular SEQ ID No. 6.

Preferably, the monoclonal antibody according to the present inventionwhich is capable to specifically recognise the E7 protein of HPV 16,preferably capable of recognising an epitope with the amino acidsequence of SEQ ID No. 3 and 7, in particular SEQ ID No. 6, comprises,preferably consists of a heavy chain having the cDNA sequence of SEQ IDNo. 9 or a light chain having the cDNA sequence of SEQ ID No. 10 orboth.

Thus, the present invention relates to a monoclonal antibody or fragmentthereof, wherein said antibody or antibody fragment comprises an aminoacid sequence coded by at least one of SEQ ID No. 9 or 10. Thisencompasses also mutated versions of SEQ ID No. 9 or 10, i.e. sequencesthat comprise additions, deletions and/or substitutions of single ormultiple nucleotides compared to SEQ ID No. 9 or 10 as long as theantibody or antibody fragment coded by the mutated sequence stilldisplays the specificity and sensitivity of the full immunoglobulin ofthe present invention.

The present invention also relates to a hybridoma cell line, preferablya mammalian, most preferred a rabbit hybridoma cell line, capable ofproducing the above-identified monoclonal antibody according to theinvention, in particular of the antibody capable to specificallyrecognising the E7 protein of HPV 16, preferably capable of recognisingan epitope of the C-terminal region of HPV 16, in particular aconformational epitope, preferably an epitope with the amino acidsequence of SEQ ID No. 3 and 7, in particular SEQ ID No. 6. The presentinvention preferably relates to the hybridoma cell line deposited asKlon 42-3-78 at the Leibniz Institute DSMZ-German Collection ofMicroorganisms and Cell Cultures on Dec. 16, 2009, which has theaccession no. DSM ACC3034. In a preferred embodiment the presentinvention also relates to the monoclonal antibody, which is obtainablefrom the supernatant of a hybridoma cell line according to the presentinvention.

The term “hybridoma cell line” as employed herein refers to a cell lineobtained by fusing myeloma cells, preferably immortalized, mammalian, inparticular rabbit, lymphoid cells with B-cells of the spleen of amammal, preferably a rabbit that has been immunized with the desiredantigen, i.e. a purified HPV E7 protein. Such an immortal hybridoma cellline is able to produce one specific and defined type of antibody whichis secreted into the cell culture medium or supernatant. The term“monoclonal” refers to the cell line from which the antibody isobtained, wherein all the cells are clones of a single parent cell. Thispermits advantageously standardized production and purificationprocedures to obtain monoclonal antibodies of the same quality.

Furthermore, such hybridoma cell lines can be grown indefinitely in thesuitable cell culture media thereby providing an infinite source of themonoclonal antibody, without batch-to-batch variations. This isparticularly advantageous for an application of monoclonal antibodies inclinical diagnostic routines, as time-consuming tests of new antibodybatches can be omitted and diagnostic methods produce constant andreliable results.

In a preferred embodiment of the present invention, the monoclonalantibody is capable of specifically recognising an epitope of theC-terminal region, preferably the zinc finger domain, of HPV 18subtypes, in particular 18, 39, 45, 59, 68 and 70. Preferably, therecognised epitope is a conformational epitope. Preferably, the epitopecomprises the amino acid sequence set forth in SEQ ID No. 4, which is acommon sequence motif present in epitopes of the HPV 18 subtypes and is:Phe Val Cys Pro Xaa Cys Ala Xaa Xaa Gln, with Xaa being any amino acid.

The epitope motif characterised by the amino acid sequence of SEQ ID No.4 represents a consensus epitope motif of the HPV 18 subtypes.

In another aspect, the present invention relates to a monoclonalanti-HPV E7 antibody, which is capable of specifically recognising anepitope of the N-terminal region of a human papillomavirus E7 protein.In a preferred embodiment, the monoclonal antibody of this aspect of thepresent invention is capable of specifically recognising an epitope ofthe N-terminal region of the E7 protein of HPV 16 subtypes, inparticular HPV types 16, 31, 33, 35, 52, 58 or of HPV 18 subtypes, inparticular HPV types 18, 39, 45, 59, 68, 70 or of both HPV 16 and 18subtypes.

The term “N-terminal region” as employed herein refers to the at most30, 35, 40, 45, 50, 55 or 60 N-terminal amino acids of the HPV E7protein.

In a particularly preferred embodiment the monoclonal antibodyspecifically recognises an epitope of the N-terminal region of the E7protein of HPV 18 subtypes, in particular HPV types 18, 39, 45, 59, 68,70, preferably of HPV 18, HPV 45 or both. Preferably, the antibodyspecifically recognising the N-terminal region of HPV 18 E7 proteinshows cross-reactivity with HPV 11, 45, 56, 58, 59 and 70. Inparticular, the antibody specifically recognising the N-terminal regionof HPV 18 E7 protein shows a high level of cross-reactivity with HPV 18,45 and 56, an intermediate level of cross-reactivity with HPV 6 and 39,and a low level of cross-reactivity with HPV 11, 33, 52, 58, and 59 withthe respective E7 proteins transiently overexpressed by U-2OS cells inan immunofluorescence assay. In an ELISA-based assay, however, it couldbe shown that the monoclonal antibody according to the present inventioncapable of specifically recognising an epitope of the N-terminal regionof the HPV 18 E7 protein only substantially crossreacts with the E7protein of HPV 45. Also, the monoclonal antibody according to thepresent invention capable of specifically recognising an epitope of theN-terminal region was able to strongly detect endogenous E7 protein ofHPV 18 positive HeLa cells, whereas no signal was obtained inuntransfected U-2OS cells or in HPV 16 positive CaSki cells, in bothimmunofluorescence and western blot experiments.

Preferably, the recognised specific N-terminal epitope comprises,preferably consists of, the amino acid sequence set forth in SEQ ID No.5 and is: Lys Ala Thr Leu Gln Asp Ile Val Leu. These amino ac-idsrepresent positions 5 to 13 of the HPV 18 E7 protein. A furtherparticularly preferred embodiment of the N-terminal epitope isrepresented by the amino acid sequence of SEQ ID No. 8 and is: Pro LysAla Thr Leu Gln Asp Ile Val Leu. These amino acids represent positions 4to 13 of the HPV 18 E7 protein.

Advantageously, it could be shown that the monoclonal anti-HPV 18 E7antibody according to the present invention is able to detect lowamounts such as 1 pg, in particular 500 fg, of HPV 18 E7 protein in anELISA-based assay.

The present invention also relates to a hybridoma cell line, preferablya mammalian, most preferred a rabbit, hybridoma cell line, capable ofproducing the monoclonal antibody capable of specifically recognising anepitope of the N-terminal region of the HPV 18 E7 protein, preferablycapable of recognising an epitope with the amino acid sequence of SEQ IDNo. 5 or 8. The present invention preferably relates to the hybridomacell line deposited as Klon 143-7-33 at the Leibniz InstituteDSMZ-German Collection of Microorganisms and Cell Cultures on Dec. 16,2009, which has the accession no. DSM ACC3035. In a preferred embodimentthe present invention also relates to the monoclonal antibody, which isobtainable from the supernatant of a hybridoma cell line according tothe present invention.

In a particularly preferred embodiment, the monoclonal antibodiesaccording to the present invention are mammalian, preferably rabbit,monoclonal antibodies (RabMabs), preferably produced by a rabbithybridoma cell line. Due to a more sophisticated immune system, rabbitsare able to produce more elaborate antibodies than mice. This advantagecombined with the possibility to create rabbit hybridoma cell linesmakes it possible to obtain high quality monoclonal antibodies.

For various applications the monoclonal antibodies according to thepresent invention may be detectably labelled themselves with aradioactive, enzymatic or fluorescent group. A variety of techniques isavailable for labelling biomolecules such as antibodies, which arewell-known to the person skilled in the art. Commonly used labelscomprise inter alia fluorochroms, like fluorescein, rhodamine, TexasRed, Cy3 or Cy5, enzymes like peroxidase, horse radish peroxidase,β-galactosidase, alkaline phosphatase or acetylcholinesterase,radioactive isotopes, digoxygenin, colloidal metals, chemi- orbioluminescent compounds. Preferably, the monoclonal antibodiesaccording to the present invention are labelled with biotin. In anotherpreferred embodiment, the antibodies of the present invention aredetected by secondary methods, like indirect immunofluorescence.Accordingly, detectably labelled secondary antibodies may be employed tospecifically detect the monoclonal antibodies according to the presentinvention.

In a preferred embodiment, the present invention also relates to adiagnostic composition, comprising the monoclonal antibodies accordingto the present invention as an active agent. Said diagnostic compositionmay, in a preferred embodiment, comprise the monoclonal antibody of thepresent invention in soluble form or liquid phase. In another preferredembodiment the present antibodies are bound, attached and/or linked to asolid support. In another preferred embodiment, the present diagnosticcomposition comprises the monoclonal antibodies according to theinvention formulated with a diagnostically acceptable carrier, diluent,buffer or storage solution. The diagnostic composition of the presentinvention comprises in a preferred embodiment the monoclonal antibodiesindividually or in combinations according to the requirements of theintended application. Preferably, said diagnostic composition is usedfor the immunological, preferably immunohistological,immunofluorescence- or ELISA-based detection of HPV E7 protein in abiological sample to enable the diagnosis of HPV infections.

The present invention also relates to a diagnostic kit, comprising themonoclonal antibodies according to the invention as a first agent andother antibodies directed against HPV E7 proteins as a second agent. Theantibodies used as a second agent are preferably polyclonal goatantibodies, capable of recognising the E7 proteins of high-risk HPV 16,18, 31, 33, 35, 39, 45, 52, 56, 58 and 59.

The diagnostic kit of the present invention comprises in a preferredembodiment the monoclonal antibodies individually or in combinationsaccording to the requirements of the intended application.

The diagnostic kit according to the present invention is preferably usedfor immunological, preferably immunohistochemical, immunofluorescence-or ELISA-based detection of HPV E7 protein, thereby enabling thediagnosis of HPV infections. Preferably, the diagnostic kit of thepresent invention further comprises optionally one or more buffers,storage solutions and/or other reagents or materials required formedical, scientific or diagnostic purposes. Furthermore, parts of thediagnostic kit can be packaged individually in vials or bottles or incombination in containers or multi-container units.

The use of the monoclonal antibodies according to the present inventionin an in vitro method, in particular an immunological, preferablyimmunohistochemical or immunofluorescence-based in vitro method or anELISA-based, preferably direct or sandwich ELISA, in vitro method isalso contemplated by the present invention. Also, the monoclonalantibodies according to the present invention might be advantageouslyused in a test strip, for example a lateral flow test, for the detectionof HPV E7 proteins.

Thus, in a preferred embodiment the present invention relates to an invitro method for the detection of HPV E7 protein, comprising i)incubating a biological sample with the monoclonal antibody according tothe present invention and ii) measuring and/or detecting HPV E7 proteinin the biological sample by measuring and/or detecting the antibodyspecifically bound to the E7 protein.

In a particularly preferred embodiment, the present invention providesan in vitro method for the immunological, preferably immunohistochemicalor immunofluorescence-based diagnosis of HPV infections, comprising a)incubating a biological sample with the monoclonal antibodies accordingto the present invention and b) measuring and/or detecting HPV E7protein, preferably the E7 protein of high-risk HPV types, in thebiological sample by measuring and/or detecting the antibodyspecifically bound to the E7 protein and thereby allowing the diagnosisof a HPV infection. Advantageously, the application of the monoclonalantibodies according to the present invention in such a diagnosticmethod allows for a highly sensitive and reliable detection of high-riskHPV infections, as the monoclonal antibodies according to the presentinvention display a very high signal-to-noise ratio with negligiblebackground. In a preferred embodiment, the monoclonal anti-HPV E7antibody capable of specifically recognising an epitope of theC-terminal or N-terminal region of the HPV E7 protein is used in thismethod. Preferably, the monoclonal antibody against the C-terminus ofHPV 16 E7 according to the present invention is used in this method,since it shows no cross-reactivity with other HPV types and a very highsensitivity.

The term “biological sample” as employed herein is understood to referto any kind of biological tissue, cells and/or organs, preferably cervixbiopsies or smears, preferably Pap Smears.

In yet another particularly preferred embodiment, the present inventionrelates to an in vitro method for the ELISA-based diagnosis of HPVinfections, comprising aa) coating a support with capture antibodiesdirected against HPV E7 proteins, bb) adding a biological sample,preferably a cell lysate, to the coated support, cc) incubating thesupport with the monoclonal detection antibody according to the presentinvention, and dd) identifying HPV E7 protein specifically bound by thecapture antibodies by measuring and/or detecting the monoclonaldetection antibody specifically bound to the E7 protein and therebyallowing the diagnosis of a HPV infection, preferably high-risk HPVinfection. In a preferred embodiment, the monoclonal anti-HPV E7antibody capable of specifically recognising the C-terminal orN-terminal region of the HPV E7 protein is used as detection antibody inthis method. Preferably, the monoclonal antibody against the N-terminusof HPV 18 E7 according to the present invention is used, preferably asdetection or capture antibody in this method. When used as detectionantibody in this method, the antibodies according to the presentinvention are preferably biotinylated, since this leads to an increasein specificity and sensitivity of the HPV E7 detection.

Preferably, polyclonal goat antibodies are used as capture antibodiesfor the ELISA-based diagnosis method, which are capable of recognisingthe E7 proteins of high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 52,56, 58 and 59. As these polyclonal goat antibodies only bind E7 proteinsof high-risk HPV types, the application of the anti-HPV 16 E7 antibodyand the anti-HPV 18 E7 antibody as detection antibodies advantageouslyallows the detection of high-risk HPV types 16, 18, 45, 56, 58, 59 and70 which together account for >95% of all cervical cancers.

As support or solid support ELISA-plates are preferably used butdifferent carriers or materials may as well be chosen by the personskilled in the art.

Cell lysates to be tested with the ELISA-based method are preferablyprepared from biological samples, preferably cervix biopsies or smears,preferably Pap Smears.

Further preferred embodiments are the subject matter of the subclaims.

The sequence listing shows:

SEQ ID No. 1 shows the amino acid sequence of a C-terminal consensusepitope motif of the E7 protein of HPV 16 subtypes and HPV 18 subtypesand

SEQ ID No. 2 shows the amino acid sequence of a C-terminal consensusepitope motif of the E7 protein of HPV 16 subtypes and

SEQ ID No. 3 shows the amino acid sequence of the C-terminalconformational epitope of the HPV 16 E7 protein, in particularcomprising positions 85 to 97 of the HPV 16 E7 protein and

SEQ ID No. 4 shows the amino acid sequence of a C-terminal consensusepitope motif of the E7 protein of HPV 18 subtypes and

SEQ ID No. 5 shows the amino acid sequence of the N-terminal epitope ofthe HPV 18 E7 protein, in particular comprising positions 5 to 13 of theHPV 18 E7 protein and

SEQ ID No. 6 shows the amino acid sequence of the C-terminalconformational epitope of the HPV 16 E7 protein comprising positions 86to 97 of the HPV 16 E7 protein and

SEQ ID No. 7 shows the amino acid sequence of the C-terminalconformational epitope of the HPV 16 E7 protein comprising positions 86to 98 of the HPV 16 E7 protein and

SEQ ID No. 8 shows the amino acid sequence of the N-terminal epitope ofthe HPV 18 E7 protein comprising positions 4 to 13 of the HPV 18 E7protein and

SEQ ID No. 9 shows the cDNA sequence of the heavy chain of the anti-HPV16 E7 antibody of the present invention and

SEQ ID No. 10 shows the cDNA sequence of the light chain of the anti-HPV16 E7 antibody of the present invention.

The present invention is further illustrated by the followingnon-limiting examples and the accompanying figures.

The figures show:

FIG. 1 shows the sensitivity of the monoclonal antibodies of the presentinvention for the detection of recombinant E7 proteins by ELISA. ELISAplates were coated with a mixture (1:1) of goat polyclonal antibodiesagainst HPV 16 E7 and HPV 18 E7. Then, recombinant E7 proteins wereadded in different concentrations. Subsequently, the monoclonal anti-HPV16 E7 (1A) and anti-HPV 18 E7 (1B) antibodies of the present inventionwere added for the detection of the recombinant proteins bound by thegoat polyclonal antibodies, followed by incubation with a correspondingsecondary antibody for visualization.

FIG. 2 shows the sensitivity of the biotinylated monoclonal antibodiesof the present invention for the detection of recombinant E7 proteins byELISA (2 A, B) and the logarithmic representation of the proteinconcentrations detected by the antibodies of the present invention (C,D). ELISA plates were coated with a mixture (1:1) of goat polyclonalantibodies against HPV 16 E7 and HPV 18 E7. Then, recombinant E7proteins were added in different concentrations. Subsequently, thebiotinylated monoclonal anti-HPV 16 E7 (2A, C) and anti-HPV 18 E7 (2B,D) antibodies of the present invention were added for detection of theE7 proteins bound by the goat polyclonal antibodies, followed byincubation with Streptavidin-Poly-HRP-conjugate for visualization. Thedetection limit is defined as mean of the background signal (twelvemeasurements) plus three times the standard deviation.

FIG. 3 shows that the biotinylated monoclonal antibodies of the pre sentinvention do not detect recombinant proteins of low-risk HPV types (6,11) in an ELISA. ELISA plates were coated with a mixture (1:1) of goatpolyclonal antibodies directed against HPV 16 E7 and HPV 18 E7. Then,different recombinant E7 proteins of high-risk HPV types (16, 18) andlow-risk HPV types (6, 11) were added. Subsequently, the biotinylatedmonoclonal anti-HPV 16 E7 (3A) and anti-HPV 18 E7 (3B) antibodies of thepresent invention were added for the detection of the E7 proteins boundby the goat polyclonal antibodies, followed by incubation withStreptavidin-Poly-HRP-conjugate for visualization.

FIG. 4 shows the detection of endogenous E7 protein of CaSki-cells (HPV16 positive) (4A) and HeLa-cells (HPV 18 positive) (4B) in thebackground of HPV negative U-2OS-cells with the biotinylated monoclonalantibodies of the present invention by ELISA. Therefore, ELISA plateswere coated with a mixture (1:1) of goat polyclonal antibodies againstHPV 16 E7 and HPV 18 E7. Cell lysates with a total cell concentration of100,000 cells per well consisting of HPV negative U-2OS-cells asbackground and different amounts of HPV 16 E7 positive CaSki-cells orHPV 18 E7 positive HeLa-cells were added. Subsequently, the biotinylatedmonoclonal anti-HPV 16 E7 (4A) and anti-HPV 18 E7 (4B) antibodies of thepresent invention were added for the detection of E7 proteins bound bythe goat polyclonal antibodies, followed by incubation withStreptavidin-Poly-HRP-conjugate for visualization. Detection limit isdefined as mean of the background signal (12 measurements) plus threetimes the standard deviation.

FIG. 5 shows the detection of E7 protein in five clinical samples (oneHPV 16 positive, one HPV 18 positive and three HPV negative) with thebiotinylated monoclonal antibodies of the present invention by ELISA.ELISA plates were coated with a mixture (1:1) of goat polyclonalantibodies against HPV 16 E7 and HPV 18 E7. Lysates of the clinicalsamples (100 μg/well) were added. Subsequently, the biotinylatedmonoclonal anti-HPV 16 E7 (5A) and anti-HPV 18 E7 (5B) antibodies of thepresent invention were added for the detection of E7 proteins bound bythe goat polyclonal antibodies, followed by incubation withStreptavidin-Poly-HRP conjugate for visualization.

FIG. 6 shows the detection of E7 protein in LBC samples form healthywomen spiked with CaSki cells (HPV 16 positive) and U-2OS cells (HPVnegative). Liquid-based cytology was prepared from a cervical smear of aPapII proband, which did not yield any signal with anti-HPV 16 E7antibody of the present invention. As indicated, 10,000 cells each ofCaSki and U-2OS were added to the sample. HPV 16 E7 positive cells werespecifically detected by immunohistochemistry with anti-HPV 16 E7antibody of the present invention over a wide range of antibodydilutions, as indicated.

FIG. 7 shows the specificity of the monoclonal antibody against HPV 16E7 employing a direct ELISA using 2.5 and 10 ng of randomly coated HPVE7 proteins of different HPV types detected by the biotinylatedmonoclonal antibody against HPV 16 E7 (14 ng/100 μl) with the buffer asa control.

FIG. 8A shows the detection of the wild-type and several mutated HPV 16E7 proteins transiently expressed in U-2OS cells by the monoclonalantibody against HPV 16 E7 (2 ng/μl) followed by alexaFlour488-labelledsecondary antibody in indirect immunofluorescence experiments.(vector=empty control vector, WT=wild-type). FIG. 8B shows the detectionof the wild-type and mutated HPV 16 E7 protein, by known polyclonal antiHPV 16 E7 antibodies which do not specifically recognise theconformational epitope in the zinc finger domain and therefore alsorecognise the mutations concerning the cystein residues.

FIG. 9 shows a model of the structure of the HPV 16 E7 protein deducedfrom the NMR structure of HPV 45 E7, containing two β-sheets (β1 and β2)and two α-helices (α1 and α2) as secondary structures. The side chainsof the four cysteins (C58, C61, C91, C94) in the two CXXC motifscoordinating zinc located in the turn connecting β1 and β2 and in theC-terminal α2-helix are indicated. The epitope 86TLGIVCPICSQK97 (SEQ IDNo. 6) in the carboxyl-terminal E7 zinc-finger recognized by themonoclonal antibody is indicated. Moreover, the localization of theC-terminal mutations Δ52YNIVT56, Δ65LRL67, Δ75DIR77, Δ79LEDLL83, C58Gand C91G are indicated. The unstructured N-terminus and the mutationsH2P and C24G are not indicated.

FIG. 10 shows epitope mapping using synthetic HPV 16 E7 13 mer peptideson microarrays which were detected by the monoclonal antibody againstHPV 16 E7, two unrelated rabbit antibodies and the secondary antibodyonly (anti-rabbit Cy5). Specifically, peptides 43 and 44 are recognisedby the monoclonal anti-HPV 16 E7 antibody which comprises the specificepitope.

FIG. 11 shows the specificity of the monoclonal antibody against HPV 18E7 employing an ELISA assay. ELISA plates were coated with a mixture(1:1) of goat polyclonal antibodies against HPV 16 E7 and HPV 18 E7.Then, 100 μg recombinant E7 protein of different HPV types was added.Subsequently, the biotinylated monoclonal anti-HPV 18 E7 antibody of thepresent invention was added for detection of the E7 proteins bound bythe goat polyclonal antibodies, followed by incubation withStreptavidin-Poly-HRP-conjugate for visualization.

FIG. 12 shows a western blot with purified E7 protein of HPV 16 and 18(10 ng) each loaded on a 12.5% SDS gel and probed with the antibodyagainst HPV 18 E7, wherein only the HPV 18 E7 protein is recognized bythe antibody (left panel) and cell extracts from HPV 18 positive HeLacells, HPV 16 positive CaSki cells and U-2OS cells transfected eitherwith an empty vector or an expression vector for HPV 18 E7. Cell lysateswere separated by SDS-PAGE and probed in western blot with the antibodyagainst HPV 18 E7 (right panel). Only the HPV 18 E7 protein isrecognised by the antibody.

FIG. 13 shows epitope mapping using synthetic HPV 18 E7 11 mer peptideswhich were detected by the monoclonal antibody against HPV 18 E7, twounrelated rabbit antibodies and the secondary antibody only (anti-rabbitCy5). Specifically, peptides 1 to 3 are recognised by the monoclonalanti-HPV 18 E7 antibody which comprise the specific epitope.

EXAMPLE 1

Generation and Purification of Rabbit Monoclonal Antibodies (RabMabs)

Purified HPV 16 and HPV 18 E7 proteins (purity >95%; 4 mg each) (Fiedleret al., J Gen Virol, 2005, 86, 3235-3241, and Fiedler et al., J VirolMethods, 2006, 134, 30-35) were used to immunize rabbits, and rabbithybridoma clones were prepared. Selected hybridomas were taken inculture and supernatants (2 I each) were produced, which typicallyyielded 2 mg of the respective RabMab. Hybridoma supernatant was dilutedwith ⅓ binding buffer (20 mM sodium phosphate, pH 7) and filteredthrough a 0.45 μm filter. The column (HiTrap™ Protein G HP 5 ml; GEHealthcare) was washed with 5-10 column volumes of binding buffer at 5ml/min. The filtered supernatant was applied and the column washed with5-10 column volumes of binding buffer; eluted with 6×2 ml elution,buffer (0.1 M glycine-HCl, pH 2.7) into collection tubes with 80 μl 3 MTris-HCl, pH 9. After elution, the column was washed with 5-10 columnvolumes of binding buffer followed by 5-10 column volumes of 20%ethanol. IgG-containing fractions were pooled and dialyzed overnight at4° C. against dialysis buffer (166 mM potassium phosphate, 83 mMglycine, pH 7.2). After dialysis, samples were concentrated usingconcentration tubes (Amicon Ultra, MWCO 10,000 Millipore). Finally, thesample was cleared by centrifugation (13,000 rpm, 4° C., 10 min) and theIgG concentration was determined using OD at 280 nm. Aliquots werestored in liquid nitrogen.

Hybridoma clones producing the desired antibodies were selected afterfurther testing and a hybridoma clone producing monoclonal anti-HPV 16E7 antibodies has been deposited on 16 Dec. 2009 with the DSMZ,Braunschweig, Germany, under the accession no. DSM ACC 3034 and a secondhybridoma clone producing monoclonal anti-HPV 18 E7 antibodies has beendeposited on 16 Dec. 2009 with the DSMZ under the accession no. DSM ACC3035.

EXAMPLE 2

Cross-Reactivity Test for E7 Antibodies

Overexpression in U-2OS Cells

U-2OS-cells (HPV negative) were transiently transfected with vectors foroverexpression of selected E7 proteins. For confocal immunofluorescencemicroscopy, cells were fixed in 4% (w/v) PFA/PBS and permeabilized with0.1% (w/v) sodium citrate/0.3% (v/v) TRITON™ X-100t-octylphenoxypolyethoxyethanol. After blocking with 1% (w/v) bovineserum albumin/PBS, cells were incubated for 1 h with the monoclonalanti-HPV 16 E7 or anti-HPV 18 E7 of the present invention (25 μg/ml) atroom temperature and analyzed by confocal microscopy.

The results showed that the anti-HPV 16 E7 antibody of the presentinvention did not cross-react with E7 proteins of other HPV types, suchas HPV 18 and HPV 45, and the anti-HPV 18 E7 antibody of the presentinvention was able to detect E7 proteins of HPV 18 and 45, 11, 56, 58,59 and 70.

Direct ELISA Assay

To more precisely characterise the cross reactivity of the monoclonalantibody against HPV 16 E7 an direct ELISA assay was used. Differentamounts of randomly coated recombinant HPV E7 proteins of different HPVtypes were analysed regarding the reactivity of the monoclonal antibodyagainst HPV 16 E7.

The assay was carried out as follows:

Wells of microtiter plates (Maxisorp F, Nunc, Vienna) were coatedovernight (4° C.) with different amounts (2.5 and 10 ng) of recombinantbacterially produced untagged HPV E7 proteins in 100 μl of coatingbuffer (0.1 M NaHCO3, pH 9.6). After washing three times in PBS, pH 7.4,containing 0.05% TWEEN®20 polysorbate20, wells were blocked with 300 μlUniversal Casein Diluent/Blocker (UCDB, SDT, Baesweiler, Germany) for 2hours at room temperature. Wells were washed three times. 100 μlbiotinylated primary monoclonal antibody against HPV 16 E7 (appropriatedilution in UCDB) was added to each well and incubated for 1 hour atroom temperature. After three washing steps, 100 μlStreptavidin-PolyHRP40 conjugate (SDT, Baesweiler, Germany, 0.2 μg/ml inUCDB) were added to each well, followed by 1 hour incubation at roomtemperature. After washing six times, successful binding of the antibodywas visualized by the addition of 100 μl chromogenic substrate(es(HS)TMB, SDT, Baesweiler, Germany) to each well and follow-upincubation for 30 min in the dark at room temperature. The reaction wasstopped by the addition of 50 μl 2N H₂SO₄ and quantified by absorbancemeasurement (450 nm) in a multilabel plate reader (VICTOR™ X5, PerkinElmer, Vienna, Austria).

As shown in FIG. 7, the monoclonal antibody against HPV 16 E7 verystrongly detected the HPV 16 E7 protein, but did not cross react withall other HPV types, including ten other high-risk HPV genotypes and thetwo most common low-risk HPV viruses, HPV 6 and HPV 11.

ELISA Assay

To more precisely characterise the cross reactivity of the monoclonalantibody against HPV 18 E7 an ELISA assay was used (see example 5 forELISA protocol). 100 μg of the respective recombinant HPV E7 proteinswere added to the wells and detected with the anti-HPV 18 E7 antibodyaccording to the present invention.

This experiment showed that the monoclonal anti-HPV 18 E7 antibodyaccording to the present invention is highly specific for the E7proteins of HPV 18 and 45 (see FIG. 11) but does not recognise the E7proteins of other high-risk HPV types.

Western Blot

The specificity of the anti-HPV 18 E7 antibody of the present inventionwas also analyzed by Western blot. Purified recombinant E7 protein ofthe HPV types 16 and 18 was separated by SDS-PAGE and analyzed byWestern blot using the anti-HPV 18 E7 antibody. No signal was obtainedfor recombinant E7 protein of HPV 16, whereas positive signals wereobtained for recombinant HPV 18 E7 (FIG. 12). Similarly, specificsignals were obtained with the anti-HPV 18 E7 antibody, when extracts oftransiently transfected U-2OS cells, expressing FLAG-tagged versions ofHPV 18 E7, were analyzed. Finally, endogenous E7 proteins of HPV 18positive HeLa cells, but not of HPV-16 positive CaSki cells, weredetectable by Western blot (FIG. 12).

EXAMPLE 3

Immunohistochemical Detection of HPV E7 Protein in Cervical CancerBiopsies

To determine the HPV status in cervical biopsies from 30 patients,first, PCR analysis was used to determine the different HPV genotypes.26 (87%) of the biopsies were HPV-DNA positive, nine (30%) containedonly HPV 16 DNA and three (10%) only HPV 18 DNA. Eight (23%) were HPV 16and 18 positive and six (23%) contained HPV 16, 18 and other HPV types.In the remaining four biopsies the HPV type was apparently notdetectable by the PCR analysis. As negative controls, 22 cervicalbiopsies containing normal, squamous and glandular epithelia were used.To test, whether the anti-HPV 16 E7 and anti-HPV 18 E7 monoclonal rabbitantibodies according to the invention could detect the HPV E7 proteinsin paraffin sections of these biopsies the following protocol forimmunohistochemistry was employed.

Immunohistochemistry was performed on paraffin-embedded tissue sectionsderived from cervical biopsies. 2 μm sections were mounted on slides,deparaffinized in xylene (2×12 minutes), rehydrated and processed forantigen retrieval by treatment for 1 hour in a steamer in TargetRetrieval Solution (DAKO S1700) for the detection of HPV E7. Endogenousperoxidase activity was blocked by incubation in 20% H₂O₂/methanol for30 minutes. Sections were washed and incubated for 15 minutes inblocking buffer (10% goat or rabbit serum from DAKOCytomation Germany,respectively, 5% BSA in 1×Tris buffer). Blocking solution was removed.The sections were either incubated with the biotinylated monoclonalrabbit anti-HPV 16 E7 or 18 E7 antibodies of the present invention (atappropriate dilutions) for 1 h at room temperature in 5% BSA/1×TBS in awet chamber. Samples were rinsed in 1×Tris/0.1% TWEEN®20 polysorbate 20and incubated with secondary IgGs (DAKOCytomation, Germany) for 45 minat room temperature in a wet chamber. After washing, samples wereincubated with streptavidin peroxidase conjugate (Sigma, Vienna) for 30minutes at room temperature. Bound antibodies were visualized with DABsolution (Sigma, Vienna) as substrate. Counterstaining was performedwith Hemalaun (Merck, Vienna). The specimens were dehydrated, andmounted using Entellan (Merck, Vienna). Brightfield microscopy withphotography was performed using Olympus CH30 microscope and a SonyDSC-W15 Cyber-shot camera.

In all 23 biopsies tested positive for HPV 16 DNA by the PCR analysis,the anti-HPV 16 E7 antibody of the present invention recognised almostall epithelial tumor cells within the tumor islets but stained no cellsin adjacent connective tissues. No HPV 16 E7 protein was detected innormal cervical specimens, neither in connective tissue and cervicalglandular epithelia nor in normal cervical squamous epithelia.

To determine whether the HPV 18 E7 protein could be detected by theanti-HPV 18 E7 antibody of the present invention immunohistochemistryexperiments were conducted using the biopsies tested positive for HPV 18DNA by the PCR analysis. Similar to the anti-HPV 16 E7 antibody, theanti-HPV 18 E7 antibody stained almost all tumor cells within therespective tumors, but did not stain cells in the adjacent connectivetissue. No staining was detected in normal cervical specimens.

Surprisingly, the anti-HPV 16 E7 as well as the anti-HPV 18 E7 antibodyof the present invention could detect the respective protein in the fourbiopsies in which HPV DNA was undetectable by the PCR analysis,suggesting that the cells of these biopsies were indeed HPV infectedwhich was not detected by the PCR analysis.

EXAMPLE 4

Detection of High-Risk HPV 16 E7 Proteins in Liquid-Based Cytology

Based on results obtained with paraffin sections, it was tested whetherthe anti-HPV 16 E7 antibody according to the present invention can beused in liquid-based cytology (LBC). To establish the procedure, LBCsamples from healthy women were spiked with CaSki cells (HPV 16positive), and U-2OS cells (HPV negative) and processed according to thefollowing protocol:

CaSki cells and U-2OS cells were harvested and resuspended inThin-Prep-Buffer (Cytyc SA, Geneva, Switzerland). Cervical swabs wereresuspended in ThinPrep-Buffer, mixed with the cultured cells, and 10 to100 μl of the mixture were dropped with a pipette on a ThinPrep-glassslide (Cytyc SA, Geneva, Switzerland). The cells were allowed to dryover night at room temperature. Cells were fixed using 4% PFA at RT for30 min in a cuvette, and washed by gentle shaking 2×5 min at RT inTBS+0.05% TWEEN®20 polysorbate20. For antigen retrieval, cells wereincubated with 10 mM NaCitrate pH 6.0+0.3% TRITON™ X-100t-octylphenoxypolyethoxyethanol for 15 min at RT in a cuvette, washed2×5 min at RT in TBS+0.05% TWEEN®20 polysorbate20. After peroxidaseblocking, slides were incubated for 30 min with 200 μl normal goat serum(DakoCytomation) in a wet chamber (1:10 dilution in 1% BSA-TBS+0.05%TWEEN®20 polysorbate20). Each slide was incubated with 200 μl primaryantibody at appropriate dilution in 1% BSA-TBS+0.05% TWEEN®20polysorbate20 for 1 hour at RT in a wet chamber. Slides were washed andincubated with biotinylated secondary antibody (DakoCytomation) atappropriate dilution in 1% BSA-TBS+0.05% TWEEN®20 polysorbate20 for 1hour at RT. Slides were washed and incubated with 200 μl PeroxidaseConjugate (Sigma; 1:500 dilution in 1% BSA-TBS+0.05% TWEEN®20polysorbate20) in a wet chamber, and subsequently rinsed in distilledwater. For visualization, peroxidase Substrate Kit SK-4200AEC-Kit(Vector, Vienna, Austria) was used. Haematoxylin served as counterstain.

This procedure yielded a very specific staining of the HPV 16 positiveCaSki cells with the anti-HPV 16 E7 antibody of the present invention,whereas neither cervical cells from healthy probands, nor U-2OS cellswere stained, suggesting a very high specificity of the antibody (seeFIG. 6).

EXAMPLE 5

ELISA-Based HPV Diagnosis

Cell Lysis

Cells from cell cultures were washed with PBS and resuspended inice-cold lysis buffer (PBS—0.1% TWEEN®20 polysorbate20, containing 1complete EDTA-free protease inhibitor cocktail tablet (Roche) per 50 mllysis buffer). Lysates were frozen at −80° C., thawed at roomtemperature, and centrifuged (4° C., 13,000 rpm, 20 min). Supernatantswere processed in the ELISA procedure.

In the case of clinical samples, cervical smears were collected from thecervix using a brush. The brush was placed immediately into thecollecting tube containing 1 ml lysis buffer without protease inhibitors(PBS-0.1% TWEEN®20 polysorbate20). After the stick of the brush wasshortened and the collecting tube was closed, the sample was storedimmediately at −80° C. until use. The sample was thawed at roomtemperature and immediately 20 μl of 50× protease inhibitor stocksolution (complete EDTA-free, Roche) per collecting tube was added.After resuspending the remaining sample on the brush by mixing it in thelysis buffer, the remaining liquid from the brush was wiped off at theedge of the tube and the brush was discarded. The sample was centrifugedat 4° C., 13,000 rpm for 20 min. Supernatants were processed in theELISA procedure.

ELISA Protocol

100 μl coating buffer (0.1 M NaHCO₃, pH 9.6) containing a mixture ofgoat polyclonal anti-high-risk HPV antibodies was added to each well ofa 96 well plate (F96 Maxisorp Nunc-Immuno Plate), and incubatedovernight at 4° C. Wells were washed 3× with washing buffer (PBS, 0.05%TWEEN®20 polysorbate20; pH 7.4); 300 μl blocking buffer (UniversalCasein Diluent/Blocker, SDT) was added to each well, and incubated for 2hours at room temperature. Wells were washed 3× with washing buffer,afterwards recombinant E7 protein or cell lysate (200 μl; diluted inlysis buffer: PBS, 0.1% TWEEN®20 polysorbate20, protease inhibitors) wasadded and incubated for 30 minutes at room temperature. Wells wereaspirated and again 200 μl protein or cell lysate were added andincubated for 30 minutes at room temperature, Wells were washed 3× withwashing buffer. 100 μl biotinylated monoclonal detection antibody(anti-HPV 16 E7 or anti-HPV 18 E7 antibodies of the present invention)in its appropriate dilution in Universal Casein Diluent/Blocker wereadded to each well, incubated for 1 hour at room temperature, and washed3× with washing buffer. 100 μl SA-PolyHRP-conjugate (SDT) in itsappropriate dilution in Universal Casein Diluent/Blocker were added toeach well and incubated for 1 hour at room temperature. Afterwards wellswere washed 6× with washing buffer. 100 μl detection reagent (es(HS)TMB,SDT) were added to each well, incubated for 30 min in the dark at roomtemperature. 50 μl stop solution (2N H₂SO₄) were added to each well, andabsorbance (450 nm) determined by an ELISA reader.

Results

To determine the sensitivity of the monoclonal detection antibodies ofthe present invention decreasing amounts of the respective recombinantE7 protein were tested with the ELISA protocol. In this setting, 10 μgof HPV 16 E7 was easily detectable by the monoclonal anti-HPV 16 E7antibody and similar sensitivity was obtained for the HPV 18 E7 antibodywith HPV 18 E7 and HPV 45 E7 proteins (see FIG. 1).

When the monoclonal detection antibodies of the present invention werebiotinylated and incubated with streptavidin-poly-HRP-conjugate insteadof a secondary antibody, an additional signal amplification could beachieved. In this optimised setting, the specific detection limit of themonoclonal antibodies according to the present invention was reduced to500 fg-1 pg in case of HPV 16 E7 and to 250 fg-500 fg for HPV 18 E7protein (see FIG. 2).

With this setting it could also be shown that proteins of low-risk HPVslike HPV 6 or HPV 11 are not detected by the anti-HPV 16 E7 or theanti-HPV 18 E7 antibody of the present invention (see FIG. 3).

To determine, whether this ELISA format is suitable for the detection ofE7 proteins in tumor cells, HPV negative cells (U-2OS) were mixed withdecreasing amounts of HeLa cells (HPV 18 positive) and CaSki cells (HPV16 positive), respectively. Cell mixtures were lysed and analyzed byELISA (see above). These experiments revealed that 2,500-5,000 CaSkicells as well as 500-1,000 HeLa cells (in the background of HPV negativeU-2OS cells) can be detected by the respective antibody of the presentinvention (see FIG. 4).

To determine, whether E7 proteins could also be detected in clinicalsamples with this setting, cervical smears were tested. For thispurpose, two consecutive Pap Smears were taken from patients or healthyprobands. One sample was used for cytological classification accordingto Papanicolaou and subsequent determination of the HPV subtype by PCRanalysis. The second sample was processed for the ELISA protocol (seeabove). In total, five samples were processed (one HPV 16 positive, oneHPV 18 positive and three HPV negative), yielding clear signals abovebackground using the antibodies according to the present invention forthe HPV 16 and 18 positive samples, whereas the HPV negative samples didnot produce a detectable signal. These data show that the sensitivity ofthe present assay employing the antibodies of the present invention ishigh enough to allow reliable detection of high-risk E7 proteins inclinical samples and thereby enabling reliable diagnosis of high-riskHPV infections (see FIG. 5).

EXAMPLE 6

Mutational Analysis of the Conformational Epitope Recognised by theMonoclonal Antibody Against HPV 16 E7

To further investigate the specificity of the monoclonal antibodyagainst HPV 16 E7 to its epitope in the C-terminal domain U-2OS cellswere transiently transfected with expression vectors for HPV 16 E7mutants addressing the unstructured N-terminus as well as the majorstructure determining elements in the C-terminal domain. Preliminarywestern blot analysis showed that the protein levels of all HPV 16 E7mutants were either similar or higher as the level of the HPV 16 E7wild-type protein. To investigate whether the monoclonal anti HPV 16 E7antibody is able to recognise the HPV 16 E7 mutants, animmunofluorescence assay was performed with the transiently transfectedU-2OS cells. The analysed mutant HPV 16 E7 proteins were: H2P (change ofhistidine residue nr. 2 to proline), C24G (change of cysteine residuenr. 24 to glycine), deletions in the β1-sheet (Δ52YNIVT56), the β2-sheet(Δ65LRL67), and the α1-helix (Δ75DIR77 and Δ79LEDLL83) as well as pointmutations in the zinc coordination site of the zinc finger domain (C58G,C91G and C58G/C91G). The positions of the mutations within the HPV 16 E7protein are also indicated in FIG. 9.

As shown in FIG. 8A the monoclonal antibody against HPV 16 E7 recognisedall deletion mutants, but was not able to recognise the point mutationsconcerning the cystein residues of the zinc coordination site. Thisindicates that the recognised epitope is located in the zinc fingerstructure and, more importantly, an intact zinc finger domain, which cannot be formed if one or more of the cystein residues is mutated, isnecessary for the efficient recognition of the HPV 16 E7 protein by themonoclonal anti HPV 16 E7 antibody. In contrast, as shown in FIG. 8B,known polyclonal antibodies against HPV 16 E7 which do not recognise thespecific conformational epitope recognised by the monoclonal antibodyagainst HPV 16 E7 also can detect the mutations concerning the cysteinresidues of the zinc finger domain.

For the indirect immunofluorescence experiments the following protocolwas used:

Cells were fixed with 4% (w/v) PFA/1×PBS, permeabilized with 0.1% (w/v)Na-Citrate/0.3% (v/v) TRITON™ X-100 t-octylphenoxypolyethoxyethanol,blocked with 1×PBS/1% BSA and incubated for 1 hour at 37° C. with theanti HPV 16 E7 antibody in 1×PBS/1% BSA. After washing in 1×PBS andstaining with alexaFlour488-labelled secondary antibody (DAKOCytomation,Hamburg), cells were processed for indirect immunofluorescencemicroscopy and viewed using a confocal laser-scanning system.

EXAMPLE 7

Epitope Mapping

HPV 16 E7 epitopes were analyzed by JPT Peptide Technologies GmbH(Berlin, Germany) using peptide microarrays. To do this, a collection of44 different HPV 16 E7 derived 13mer peptides, positioned on peptidemicroarrays, were incubated with the monoclonal antibody against HPV 16E7 and unrelated rabbit control antibodies. The determination ofpeptide-antibody binding was performed by RepliTope-analysis where thepeptide microarray was incubated with the primary antibody followed by afluorescently labelled secondary antibody (anti-rabbit-Cy5). Afterwashing the peptide microarrays were dried using a microarray centrifugeand scanned in a high resolution microarray scanning system withappropriate wavelength settings.

As shown in FIG. 10, peptides 43 and 44 which comprise amino acids 85 to97 (SEQ ID No. 3) and 86 to 98 (SEQ ID No. 7), respectively, arespecifically recognised by the monoclonal antibody against HPV 16 E7,indicating that these amino acids represent the recognised epitope.

Similarly, a collection of 47 different HPV 18 E7 derived overlapping 11mer peptides was used for epitope mapping for the anti-HPV 18 E7antibody according to the present invention. The peptide microarrayswere incubated with the anti-HPV 18 E7 antibody as well as two unrelatedmonoclonal antibodies.

As shown in FIG. 13, peptides 1 to 3 which comprise the N-terminal aminoacids 4 to 13 of the HPV 18 E7 protein are specifically recognized bythe anti-HPV 18 E7 antibody of the present invention.

The invention claimed is:
 1. A monoclonal anti-HPV (humanpapillomavirus) E7 antibody, which is capable of specificallyrecognising an epitope of the C-terminal region of an HPV 16 E7 protein,wherein the epitope comprises the amino acid sequence set forth in SEQID No.
 6. 2. The monoclonal antibody according to claim 1, wherein theepitope is an epitope of a zinc finger domain of the C-terminal regionof the HPV E7 protein.
 3. The monoclonal antibody according to claim 1,wherein the epitope is a conformational epitope.
 4. The monoclonalantibody according to claim 1, wherein the antibody is labelled with aradioactive, enzymatic or fluorescent group.
 5. A diagnosticcomposition, comprising a monoclonal antibody according to claim 1 as anactive agent.
 6. A diagnostic kit, comprising a monoclonal antibodyaccording to claim 1 as a first agent and antibodies directed againstHPV E7 proteins as a second agent.
 7. A monoclonal anti-HPV (humanpapillomavirus) E7 antibody, which is capable of specificallyrecognising an epitope of the N-terminal region of an HPV 18 E7 protein,wherein the epitope comprises the amino acid sequence set forth in SEQID No.
 8. 8. The monoclonal anti-HPV E7 antibody according to claim 7,wherein the recognised HPV E7 protein is the E7 protein of HPV
 45. 9.The monoclonal antibody according to claim 7, wherein the antibody islabelled with a radioactive, enzymatic or fluorescent group.
 10. Adiagnostic composition, comprising a monoclonal antibody according toclaim 7 as an active agent.
 11. A diagnostic kit, comprising amonoclonal antibody according to claim 7 as a first agent and antibodiesdirected against HPV E7 proteins as a second agent.
 12. An in vitromethod for detection of an HPV E7 protein from HPV 16 or HPV 18 or HPV45, the method comprising: i) incubating a biological sample with atleast one monoclonal antibody selected from a group consisting of: amonoclonal anti-HPV (human papillomavirus) E7 antibody, which is capableof specifically recognising an epitope of the C-terminal region of anHPV 16 E7 protein, wherein the epitope comprises the amino acid sequenceset forth in SEQ ID No. 8, and a monoclonal anti-HPV (humanpapillomavirus) E7 antibody, which is capable of specificallyrecognising an epitope of the N-terminal region of an HPV 18 E7 protein,wherein the epitope comprises the amino acid sequence set forth in SEQID No. 8; and ii) measuring and/or detecting the HPV E7 protein in thebiological sample by measuring and/or detecting the monoclonal antibodyspecifically bound to the E7 protein.
 13. The method according to claim12, wherein measuring and/or detecting HPV E7 protein in the biologicalsample includes detecting HPV 16 or HPV 18 or HPV 45 E7 protein in thebiological sample by detecting the antibody specifically bound to theHPV 16 or HPV 18 or HPV 45 protein and thereby allowing a diagnosis of aHPV infection.
 14. An in vitro method for diagnosis of HPV infections,the method comprising: a) coating a support with capture antibodiesdirected against HPV 16, HPV 18 or HPV 45 E7 proteins; b) adding abiological sample to the coated support; c) incubating the support witha monoclonal detection antibody, wherein the monoclonal detectionantibody is at least one monoclonal antibody selected from a groupconsisting of: a monoclonal anti-HPV (human papillomavirus) E7 antibody,which is capable of specifically recognising an epitope of theC-terminal region of an HPV 16 E7 protein, wherein the epitope comprisesthe amino acid sequence set forth in SEQ ID No. 6, and a monoclonalanti-HPV (human papillomavirus) E7 antibody, which is capable ofspecifically recognising an epitope of the N-terminal region of an HPV18 E7 protein, wherein the epitope comprises the amino acid sequence setforth in SEQ ID No. 8; and d) identifying HPV 16 or HPV 18 or HPV 45 E7protein specifically bound by the capture antibody by detecting themonoclonal detection antibody specifically bound to the HPV 16 or HPV 18or HPV 45 E7 protein and thereby allowing a diagnosis of a HPVinfection.
 15. The method according to claim 14, wherein the method isELISA-based.
 16. An in vitro method for diagnosis of HPV infections, themethod comprising: a) coating a support with a monoclonal captureantibody, wherein the monoclonal capture antibody is at least onemonoclonal antibody selected from a group consisting of: a monoclonalanti-HPV (human papillomavirus) E7 antibody, which is capable ofspecifically recognising an epitope of the C-terminal region of an HPV16 E7 protein, wherein the epitope comprises the amino acid sequence setforth in SEQ ID No. 6, and a monoclonal anti-HPV (human papillomavirus)E7 antibody, which is capable of specifically recognising an epitope ofthe N-terminal region of an HPV 18 E7 protein, wherein the epitopecomprises the amino acid sequence set forth in SEQ ID No. 8; b) adding abiological sample to the coated support; c) incubating the support withdetection antibodies directed against HPV 16, HPV 18 E7 or HPV 45proteins; and d) identifying HPV 16 or HPV 18 or HPV 45 E7 proteinspecifically bound by the capture antibody by detecting the monoclonaldetection antibody specifically bound to the HPV 16 or HPV 18 or HPV 45E7 protein and thereby allowing a diagnosis of a HPV infection.
 17. Themethod according to claim 16, wherein the method is ELISA-based.
 18. Themonoclonal antibody according to claim 1, wherein the monoclonalanti-HPV antibody is made by: immunizing a rabbit with purified HPV 16E7 protein; isolating B-cells from a spleen of the immunized rabbit;fusing the B-cells with myeloma cells to generate hybridoma cells;expressing monoclonal antibodies from the hybridoma cells; isolating thehybridoma cells that express the monoclonal anti-HPV E7 antibody thatspecifically recognizes the HPV 16 E7 epitope comprising the amino acidsequence set forth in SEQ ID No. 6; and expressing the monoclonalanti-HPV antibody from the isolated hybridoma cells.
 19. The monoclonalanti-HPV E7 antibody according to claim 7, wherein the monoclonalanti-HPV antibody is made by: immunizing a rabbit with purified HPV 18E7 protein; isolating B-cells from a spleen of the immunized rabbit;fusing the B-cells with myeloma cells to generate hybridoma cells;expressing monoclonal antibodies from the hybridoma cells; isolating thehybridoma cells that express the monoclonal anti-HPV E7 antibody thatspecifically recognizes the HPV 18 E7 epitope comprising the amino acidsequence set forth in SEQ ID No. 8; and expressing the monoclonalanti-HPV antibody from the isolated hybridoma cells.